Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms

A retrospective study was performed on natural cases of postweaning multisystemic wasting syndrome (PMWS), recorded from January 1999 to December 2000, to determine the prevalence, microscopic lesions, and other coexisting pathogens associated with PMWS. PMWS is diagnosed based on three criteria: the presence of clinical signs (retardation of growth), characteristic microscopic lesions (granulomatous inflammation and inclusion body), and the presence of porcine circovirus (PCV)-2 within these lesions. One hundred and thirty three (8.1%) of the 1634 pigs submitted from 1243 pig farms were diagnosed for PMWS. The affected pigs were from 25 to 120 days old, the majority (78 cases, 58.6%) being 60 to 80 days old. PMWS occurred each month during the two-year study period, but the incidence peaked in May (38 cases, 28.6%), followed by April (18 cases, 13.5%) and June (13 cases, 9.8%). The most consistent and characteristic lesions were multifocal, granulomatous inflammation in lymph nodes, liver and spleen, characterized by infiltration of epithelioid macrophages and multinucleated giant cells. The majority of cases (113 cases, 85.0%) was dual infection with other pathogens. The combination of PCV-2 and Hemophilus parasuis (43 cases, 32.3%) was shown to be the most prevalent followed by PCV-2 and porcine reproductive and respiratory syndrome virus (39 cases, 29.3%). The consistent presence of PCV-2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PMWS, has led to the speculation that PCV-2 is the etiological agent causing PMWS.     

Effects of the timing of the administration of Mycoplasma hyopneumoniae bacterin on the development of lesions associated with porcine circovirus type 2

To determine whether there is an effect of the timing of vaccination on porcine circovirus type 2 (PCV-2) replication and PCV-2-associated lesions, 78 pigs were randomly assigned to eight groups: group 1 (10 pigs) was vaccinated with a commercial Mycoplasma hyopneumoniae vaccine at two and four weeks of age, group 2 (nine pigs) was vaccinated at four and six weeks of age, group 3 (10 pigs) at six and eight weeks of age and group 4 (10 pigs) at eight and 10 weeks of age; group 5 (nine pigs) was vaccinated once with a double dose at four weeks of age, and group 6 (10 pigs) was vaccinated once with a double dose at eight weeks of age. Groups 7 and 8, both of 10 pigs, were not vaccinated. At eight weeks of age, the pigs in groups 1 to 7 were inoculated with PCV-2. Fourteen days after they had been inoculated, the pigs in groups 1, 4 and 5 had significantly (P<0.05) more copies of the PCV-2 genome in their serum than the unvaccinated pigs. Microscopically, 14 of the 68 inoculated pigs had normal lymphoid tissues, 40 had mild PCV-2-associated lymphoid lesions and 14 had moderate lesions. The mean overall lymphoid lesions (lymphoid depletion, granulomatous inflammation, and quantity of PCV-2 antigen in spleen, tonsil, and five lymph nodes) were significantly (P<0.05) more severe in groups 4 and 5 than in groups 2, 3, 7 and 8.     

Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction.

This report describes the first diagnosis of porcine circovirus (PCV) infection in weaned pigs with postweaning multisystemic wasting syndrome in Korea by immunohistochemistry and polymerase chain reaction. The most unique lesions were multifocal granulomatous inflammation affecting lymph nodes, liver, and spleen, characterized by infiltrates of epithelioid macrophages and multinucleated giant cells. Circoviral antigen was detected in formalin-fixed sections and was usually present in large, round, dendritic cells in the white pulp of spleen and remnants of follicles in lymph nodes. Lymphoid follicles in the tonsils also contained PCV antigen. A 530-bp DNA fragment of circovirus was successfully amplified from all tested lymph nodes, liver, and spleen.     

Retrospective study of porcine circovirus 2 infection in Japan: seven cases in 1989

Retrospectively, we demonstrated that seven pigs necropsied in 1989 had characteristic porcine circovirus 2 (PCV-2) lymphoid tissue lesions of severe lymphoid depletion, syncytial giant cell formation, and intracytoplasmic inclusion bodies in macrophages. Immunohistochemically, PCV-2 antigen was detected in these tissues and the distribution of positive staining corresponded to the distribution of inclusion bodies. Electron microscopy demonstrated viral particles compatible with porcine PCV-2. Therefore, the disease occurred in Japan as early as 1989.     

猪皮炎肾病综合征组织病理学和PCR诊断

应用病理学和PCR方法,对浙江某猪场送检的疑似猪皮炎肾病综合征（PDNS）病猪进行诊断。结果发现肾脏呈现纤维蛋白性肾小球肾炎变化,真皮及皮下血管表现为坏死性脉管炎,淋巴组织中大量淋巴细胞缺失、多核巨细胞浸润,PCR扩增产物显示猪圆环病毒2型（PCV-2）阳性。结果表明,该猪场感染了PCV-2,并表现为PDNS的病理特征。     

The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease — 2004–2006

In the late fall of 2004 more severe lesions of porcine circovirus-2 associated disease (PCVAD) than usual occurred during an outbreak of porcine circovirus-2 (PCV-2) infection in Ontario nursery and grower/finisher pigs. The lesions were of unprecedented severity and included diffuse bronchointerstitial pneumonia, granulomatous enteritis, vasculitis, interstitial nephritis, and new lesions of splenic infarction. Some affected herds had up to 50% mortality. The outbreak correlated with the sudden emergence of a variant PCV-2, with PCR restriction fragment length polymorphism (RFLP) type 321. Phylogenetic comparison of ORF2 sequences and full genome sequences showed the new variant to be different from the previously dominant RFLP type 422 viruses, and similar to viruses that had occurred in France and other European and Asian countries. A subsequent retrospective study showed a statistically significant increase in the frequency of histological lesions in lymph node, spleen, lung, small intestine, colon and kidney, for pigs spontaneously infected with RFLP type 321, compared with the older RFLP type 422 strain. Viral burden, based on IHC staining in lymph node, also showed a statistically significant increase in pigs infected with the newer variant RFLP type 321, compared with the older RFLP type 422 strain. This enhanced virulence in pigs infected with PCV-2 RFLP type 321 strain may be related to the genetic differences in this new strain of PCV-2. This virus is now the dominant strain of PCV-2 virus found in Ontario and Quebec swine.     

Porcine proliferative enteropathy in Estonian pig herds: histopathology and detection of Lawsonia intracellularis by PCR

Lawsonia intracellularis is the causative agent of proliferative enteritis in pigs (PPE). This bacterium is difficult to culture from clinical samples and antemortem demonstration is therefore usually performed by PCR on faecal samples. The aim of this study was to elucidate the frequency of L. intracellularis infection in pig herds in Estonia using PCR, histopathological methods and electronmicroscopical studies. The frequency of demonstration of L. intracellularis was highest in 9-12 weeks old pigs (68.1%). It was more frequent in growing pigs with enteritis on small farms where the system of "all-in all-out" was not practiced and where standards of hygiene were poor. Gross and histopathological studies demonstrated that characteristic macroscopic changes associated with PPE were localised to the distal jejunum and ileum.Thickened longitudinal and circumferential folds occurred in the mucosa of the affected regions of the bowel. Samples from pigs aged 4 to 20 weeks exhibited the most intensive inflammatory changes. The distal part of the jejunum, ileum and the upper third of proximal colon and cecum wall were visibly thickened with reduced luminal diameter. Hyperplasia of lymphoid tissue and, in many cases, pseudomembranous or fibrinous inflammation was found. L. intracellularis was detected in 56 young pigs using histopathological methods. Additionally, in 8 of these pigs intracellular bacteria were demonstrated in ilial epithelial cells by transmission electronmicroscopical (TEM) investigation. On the basis of these TEM investigations it was concluded that L. intracellularis causes disturbances of normal growth, differentiation and apoptosis of the epithelial cells of ileum.     

Primary infection protects pigs against re-infection with Lawsonia intracellularis in experimental challenge studies

In two separate trials pigs were experimentally infected with Lawsonia intracellularis at 5-6 weeks of age followed by antibiotic treatment and resolution of the primary infection and then re-inoculated at 12-13 weeks of age. A treatment-control group of pigs received the primary infection and antibiotic treatment only, and served as control for the antibiotic treatment of the primary infection. A challenge-control group of pigs received the second inoculation dose only at 12-13 weeks of age to control infectivity of the challenge-dose and susceptibility of pigs to L. intracellularis at this age. Pigs were monitored for shedding of L. intracellularis in faeces by PCR, and for the development of antibodies and responses of acute phase proteins in serum. The presence of L. intracellularis antigen in the intestinal mucosa was examined in post mortem samples by immunohistochemistry. In both trials primary infected pigs were protected from infection after challenge inoculation as evidenced by absence of faecal shedding of L. intracellularis, lack of changes in acute phase protein concentrations after challenge and with low levels of bacterial antigen in the intestinal mucosa of re-inoculated pigs comparable to that of the treatment-control pigs. In contrast, challenge-control pigs shed L. intracellularis in faeces, had L. intracellularis antigen extensively present within all layers of the intestinal mucosa and developed a significant acute phase protein response in serum after the experimental infection. The acute phase protein response to L. intracellularis infection was detected as an increased rise in the serum concentrations of C-reactive protein and haptoglobin from day-6 post infection, and increased serum concentrations of haptoglobin were generally seen 2-3 weeks after inoculation both at 5-6 and 12-13 weeks of age. In conclusion substantial protection against L. intracellularis infection was found in the re-inoculated pigs in contrast to the development of infection in age-matched control pigs. The acute phase protein responses reflected both the observed protection against L. intracellularis infection upon secondary challenge and that increased resistance to the infection develops with age.     

Colonisation and shedding of Lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms

Lawsonia intracellularis is an intracellular bacterium causing proliferative enteropathy in various animal species, and is considered an economically important pathogen of pigs. Rats and mice have been implicated as external vectors for a wide range of pig pathogens, including L. intracellularis. Previous studies have demonstrated L. intracellularis infection and proliferative enteropathy in rodents, but did not show the duration of shedding or the number of L. intracellularis shed by infected rodents, and therefore the infection risk that rodents pose to pigs. In this study, the number of L. intracellularis shed in the faeces and intestinal mucosa of wild rats trapped on pig farms was determined by a quantitative real time polymerase chain reaction assay. The prevalence of L. intracellularis in wild rats trapped on pig farms with endemic proliferative enteropathy (PE) was very high (≥ 70.6%), and large numbers of L. intracellularis were shed (10(10)/g of faeces) in a small proportion of wild rats. The duration of colonisation in laboratory rats and mice challenged with porcine isolates of L. intracellularis was also shown. Faecal shedding of L. intracellularis persisted for 14-21 days in rats and mice that were mildly affected with histological lesions of PE. The humoral immune response to L. intracellularis persisted for 40 days in both species. This study demonstrates that rodents may be an important reservoir of L. intracellularis on piggeries, and hence rodent control is important in disease eradication programs on pig farms.     

Distribution of porcine circovirus 2 cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Ba?ka County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Ba?ka County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyer's patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.     

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting syndrome (PMWS), and to evaluate the diagnostic techniques for detecting porcine circovirus type 2 (PCV-2) and other microorganisms. Four pigs in the early stage and 11 pigs in the late stage of the disease, and eight clinically normal pigs were necropsied. Samples of lymphoid tissue and serum were also obtained from 12 slaughter pigs from the same farm. The tissues were examined histopathologically, and in situ hybridisation, serology and PCR were used to detect porcine circovirus type 1 (PCV-1) and/or PCV-2 in tissues and/or sera. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) were also investigated. Characteristic microscopical lesions of PMWS were observed in the lymphoid tissues of the pigs in all three necropsied groups; the lesions were most common and severe in the pigs in the early stage of the disease, less so in the pigs in the late stage of the disease, and least in the clinically normal pigs. PCV-2 infection was detected in all the necropsied pigs by in situ hybridisation and PCR. Only three pigs had the PCV-1 genome in serum or lymph node tissue. In contrast, the slaughter pigs had no microscopical lesions and no PCV-2 nucleic acid in their serum or tissues, and only one of them had the pCV-1 genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.     

Enteritis associated with porcine circovirus 2 in pigs

This report describes the diagnosis of porcine circovirus 2 (PCV2)-associated enteritis in 6 weaned pigs without postweaning multisystemic wasting syndrome by histopathology, virus isolation, and in situ hybridization. The most unique lesions were granulomatous inflammation affecting Peyer's patches, characterized by infiltrates of epithelioid macrophages and giant multinucleated cells. Large, multiple, basophilic or amphophilic, grape-like intracytoplasmic inclusion bodies were often seen in the cytoplasm of histiocytic cells and giant multinucleated cells. No microscopic lesions were observed in the lymphoid tissue, such as lymph node, spleen, and tonsil. A strong hybridization signal for PCV2 was detected in the cytoplasm of histiocytes and giant multinucleated cells in Peyer's patches. Porcine circovirus 2 was isolated from homogenates of the small and large intestines in 2 weaned pigs. The presence of diarrhea and granulomatous enteritis, and abundant PCV2 DNA associated with the microscopic lesions is suggestive of PCV2-associated enteritis. Thus, PCV2-associated enteritis could be a distinct clinical manifestation of PCV2.     

Detection of Lawsonia intracellularis in the tonsils of pigs with proliferative enteropathy

The presence of Lawsonia intracellularis, the obligate intracellular bacterium causing proliferative enteropathy (PE), in the tonsils of pigs as a locus for infection or extraintestinal occurrence of the bacterium was investigated by PCR and immunohistochemistry. Tonsillar occurrence of L. intracellularis could be part of the pathogenesis of PE and an important risk factor in the spread of the disease. L. intracellularis was detected by only PCR in the tonsils of 2/32 pigs without PE at necropsy but with a clinical history of diarrhoea and detection of the bacterium in faeces 1 to 3 weeks prior to necropsy but not in four pigs with moderate PE lesions. However, L. intracellularis was detected in the tonsils of 4/9 pigs with PE complicated with necroses and in 4/4 pigs with proliferative haemorrhagic enteropathy in which L. intracellularis antigen also was demonstrated in tonsillar macrophages and as intact bacteria in the lumen of the crypts. The results show that L. intracellularis is detectable in the tonsils of pigs and that the tonsillar presence of L. intracellularis appears to be correlated to the severity of the intestinal lesions possibly as a result of local retention and not as part of the pathogenesis of PE.     

Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus

One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

The microbiota of pigs influenced by diet texture and severity of Lawsonia intracellularis infection

Pigs with and without naturally occurring Lawsonia intracellularis infection were fed diets with different texture. In a previous study from 79 pig herds using a similar feeding on pelleted or non-pelleted form showed that the non-pelleted diet was associated with a reduced prevalence of L. intracellularis. In this study a mechanistic approach was taken for explaining and testing this observation by studying the microbiota and the occurrence of L. intracellularis in the distal ileum of 54 pigs by terminal restriction fragment length polymorphism (T-RFLP) analysis, Real-Time PCR and in situ hybridization. The texture of the diet influenced the microbiota, and from a quantitative discriminative analysis of the terminal restriction fragments (T-RFs) of ileum samples it was deduced that Clostridium spp. and Lactobacillus spp. were associated with the non-pelleted diet and Streptococcus spp. with the pelleted diet. In experimentally infected pigs it was verified that 89bp and 90bp sized T-RFs (HhaI) from ileum represented L. intracellularis. The non-pelleted diet seemed to reduce the relative amount of L. intracellularis in the total microbiota of the ileum, but the number of pigs detected positive with L. intracellularis by Real-Time PCR was not influenced. The five pigs with highest L. intracellularis content showed T-RFs that were not present in profiles from less or non-infected pigs, which may indicate that some bacterial species were associated with L. intracellularis infection.     

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.